Recent development of the polymerase chain reaction (PCR) has provided an important tool for the detection of nucleic acid sequences present at low concentrations (Mullis, K. B. et al., U.S. Pat. No. 4,683,195 and 4,683,202). In PCR, a segment of target sequence having boundaries defined by two oligonucleotide extension primers, is amplified through repeated enzymatic cycles to provide additional templates for further amplification reactions. Accordingly, a small number of target sequences can be exponentially amplified and readily detected. A major limitation of PCR lies in the extensive generation of by-products produced as a result of non-specific priming events, e.g., random priming of the nucleic acid template and/or self priming of the extension primers. Thus, when a high number of amplification cycles are required to amplify a target sequence present at a relatively low concentration, the products of non-specific priming events significantly impede PCR sensitivity.
An additional, related limitation of PCR is the requirement for a separation step prior to detection of the amplified target. According to standard PCR conditions, separation of the amplified target sequence from the products of non-specific priming events is a prerequisite for detection of the amplified target sequence. The absence of a homogenous amplification reaction, i.e., a reaction in which amplification and detection take place in the same reaction vessel has been an obstacle in automating the PCR procedure. In addition, the requirement for a separation step also subjects the PCR mixture to potential contamination resulting from the separation procedure. The likelihood of contamination severely limits the potential application of PCR in routine clinical diagnosis.
Attempts have been reported to develop a homogeneous assay for amplification and detection. One such attempt is described in the procedure known as the ligase chain reaction (LCR, Beckman, K. C. and Wang, C. N., European Pat. No. 320,308). LCR is performed using two pairs of immediately adjacent and ligatable probes. The probes are amplified through repeated cycles of ligation. However, the probes can randomly ligate to each other to produce a background signal which is difficult to eliminate, thus reducing the sensitivity of detection.